Use of synthetic peptides to delineate discontinuous sequence regions involved in epitope sites of the thyrotropin β-subunit

In this investigation, an overlapping set of synthetic peptides spanning the entire primary structures of the β-subunit of bovine and human thyrotropin, bTSHβ and hTSHβ respectively, have been prepared to aid the delineation of the amino acid sequence regions involved in two spatially related epitopes of bTSH. These peptides were then evaluated for their ability to inhibit the binding of two anti-hTSH monoclonal antibodies, designated mAb279 and mAb299, to radiolabeled I¹²⁵-bTSHβ using competitive radioimmunoassay procedures. Synthetic peptides related to the sequence region b/hTSHβ [56-68] were found to specifically inhibit the binding of I ¹²⁵-bTSHβ to mAb299, whilst having no effect on the binding of mAb279. In previous studies we have shown that mAb279 and mAb299 recognise epitopic sites located within the receptor-binding site of the TSH β-subunit. This investigation has therefore permitted identification of a contribution to the receptor binding site from the TSHβ [56-68] sequence, which forms part of the L3 loop region of the TSH β-subunit that is held in close proximity to the L1 loop region and the C-terminus of the TSH β-subunit by the disulphide bonds TSHβ[Cys¹⁶-Cys ⁶⁷] and TSHβ[Cys¹⁹-Cys¹⁰⁵]. This finding is in agreement with previous investigations which have shown that TSHβ [Tyr⁵⁹] and TSHβ [Tyr⁷⁴] are also associated with the mAb299 epitope site, as well as contributing to the receptor binding region of the TSH β-subunit.